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  Section: Practical Skills in Chemistry » Instrumental techniques
 
 
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Interpreting chromatograms

 
     
 
Content
Instrumental techniques
  Basic spectroscopy
    Introduction to spectroscopy
    UV Ivisible spectrophotometry
    Fluorescence
    Fluorescence spectrophotometry
    Phosphorescence and luminescence
    Atomic spectroscopy
  Atomic spectroscopy
    Atomic Absorption Spectroscopy
    Atomic Emission Spectroscopy
    Inductively coupled plasma
    Decomposition techniques for solid inorganic samples
  Infrared spectroscopy
  Nuclear magnetic resonance spectrometry
    1H-NMR spectra
    13C-NMR spectra
  Mass spectrometry
    Interfacing mass spectrometry
  Chromatography ~ introduction
    The chromatogram
    Resolution
    Detectors
  Gas and liquid chromatography
    Gas chromatography
    Liquid chromatography
    High-performance liquid chromatography
    Interpreting chromatograms
    Optimizing chromatographic separations
    Quantitative analysis
  Electrophoresis
    The supporting medium
    Capillary electrophoresis
    Capillary zone electrophoresis (CZE)
    Micellar electrokinetic chromatography (MEKC)
  Electroanalytical techniques
    Potentiometry and ion-selective electrodes
    Voltammetric methods
    Oxygen electrodes
    Coulometric methods
    Cyclic voltammetry
  Radioactive isotopes and their uses
    Radioactive decay
    Measuring radioactivity
    Chemical applications for radioactive isotopes
    Working practices when using radioactive isotopes
  Thermal analysis
    Thermogravimetry
    Applications

Make sure you know the direction of the horizontal axis of the chromatogram (usually, either volume or time) - it may run from right to left or vice versa - and make a note of the detector sensitivity on the vertical axis. Ideally, the base line should be 'flat' between peaks, but it may drift up or down owing to a number of factors including:
  • changes in the composition of the mobile phase (e.g. in gradient elution);
  • tailing of material from previous peaks;
  • carry-over of material from previous samples; this can be avoided by efficient cleaning of columns between runs - allow sufficient time for the previous sample to pass through the column before you introduce the next sample;
  • loss of the stationary phase from the column (column 'bleed'), caused by extreme elution conditions;
  • air bubbles (in liquid chromatography); if the buffers used in the mobile phase are not effectively degassed, air bubbles may build up in the flow cell of the detector, leading to a gradual upward drift of the base line, followed by a sharp fall when the accumulated air is released. Small air bubbles that do not become trapped may give spurious small peaks as they pass through the detector.
Peak close to the origin may be due to non-retained sample molecules, flowing at the same rate as the mobile phase, or to artefacts, e.g. air (GC) or solvent (HPLC) in the sample. Whatever its origin, this peak can be used to measure the void volume and dead time of the column. No peaks from genuine sample components should appear before this type of peak.

Peaks can be denoted on the basis of their elution volume (used mainly in liquid chromatography) or their retention times (mainly in GC). If the peaks are not narrow and symmetrical, they may contain more than one component. Where peaks are more curved on the trailing side compared with the leading side, this may indicate too great an association between the component and the stationary phase, or overloading of the column.

 
     
 
 
     



     
 
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